Repetitive DNA sequence specific for mycobacterium tuberculosis to be used for the diagnosis of tuberculosis

ABSTRACT

A novel composition and/or methods for the diagnosis of tuberculosis wherein the composition comprises a repetitive DNA segment that is specific for members of the Mycobacterium tuberculosis complex. The DNA segment repeats in the chromosome of Mycobacterium tuberculosis complex, and is conserved in all copies of the chromosomes. A method comprises using an entire repetitive DNA sequence, or any part thereof, as a hybridization probe for the direct detection of Mycobacterium tuberculosis complex in clinical material. In another method, a smaller portion of an entire repetitive DNA sequence is amplified using polymerase chain reaction, yielding a 123 base-pair product.

This is a division, of application Ser. No. 589,819, filed Sep. 28,1990.

FIELD OF THE INVENTION

The present invention relates to a specific DNA sequence. Moreprecisely, the present invention relates to a composition comprising aspecific DNA sequence that is unique to a pathogenic microorganism. Thepresent invention further relates to the utilization of the compositionto detect the pathogenic microorganism in clinical material.

BACKGROUND OF THE INVENTION

Tuberculosis, an infectious disease caused by the microorganismMycobacterium tuberculosis, continues to remain a major global healthproblem. In the United States, tuberculosis still persists as asignificant health problem, particularly in underprivileged and minoritypopulations, among immigrants from high-risk countries, and in otherhigh-risk groups, such as individuals with human immunodeficiency viralinfections.

A definitive diagnosis of tuberculosis depends upon the isolation ofMycobacterium tuberculosis (sometimes referred to hereinafter as "M.tuberculosis") from the secretions or tissues of an infected individualin conjunction with clinical findings of the disease. Because of thelength of time required for isolation of M. tuberculosis and thesubsequent diagnosis of tuberculosis, a long-standing goal ofresearchers has been to develop a rapid, sensitive, and specific testfor the detection of the organism in clinical specimens. Such a testcould substantially decrease the time required to definitively diagnosetuberculosis, and assist the health care provider in administering theappropriate therapeutic treatment.

A variety of methods have been used for detection of M. tuberculosis.With culture identification procedures, a specimen is typically placedon an acceptable culture medium, incubated at a specific temperature fora period of time, and then inspected for growth of the organism.Although routinely performed, these procedures are highly technical,expensive, and laborious.

A second method, direct microscopy, involves the direct examination ofsmears prepared from a clinical specimen. Although the most rapid methodfor detecting mycobacteria, direct microscopy is limiting becausetechnical expertise is required in interpreting smears and a largenumber of bacteria must be present for detection.

Non-cultural methods, such as radioimmunoassay, latex agglutination andenzyme immunosorbent assay, have also been employed for the directdetection of M, tuberculosis. These approaches appear to be promisingbecause of their rapidity. However, a major limitation with thesemethods is the lack of sensitivity in detecting the organism.

A more promising diagnostic approach has been achieved with recombinantDNA and hybridization techniques. Nucleic acid hybridization assays havebeen used to detect and identify target genetic materials such as DNA inclinical specimens. See Tenover, Clin. Microbiol. Rev. 1: 82-101, 1988.The assay is premised upon the presence of a specific nucleotidesequence in the target genetic material, and the detection of thissequence. The direct detection of the specific nucleotide sequenceactually occurs by use of a nucleic acid probe. A probe is a nucleotidesequence that is complementary to the DNA sequence of one of the strandsof the DNA molecule that is desired to be detected in the clinicalsample. A detectable marker is attached to the probe.

DNA probe technology offers advantages over other detection methods. Thedetection time is shorter, and the technology is not dependent upon theviability of the organism. Despite these benefits, a significantlimitation of this technology is its lack of sensitivity in detecting M.tuberculosis.

A DNA probe system is available for detecting species of mycobacteria,but only after the organism has grown in culture (Gen-Probe, Inc.).Gonzalez et al., Diagn. Microbiol. Infect. Dis., 8: 69-77, 1987.However, no direct probe for the detection of mycobacteria in clinicalspecimens is currently available.

SUMMARY OF THE INVENTION

The present invention provides a composition that comprises a repetitiveDNA segment that is specific for members of the Mycobacteriumtuberculosis complex (e.g., M. tuberculosis, M. bovis, and M. bovisBCG). The DNA segment can be used as a hybridization probe and as atarget of amplification for the direct detection of the DNA from theMycobacterium tuberculosis complex in clinical material.

In one embodiment of the present invention, the composition comprising aDNA segment repeats in the chromosome of M. tuberculosis.

In another embodiment, the nucleotide sequence of the DNA segment isconserved in all copies of the chromosomes of M. tuberculosis complex.

In an embodiment, the repetitive DNA segment is present in clones, λKE55and λKE58.

In another embodiment of the present invention, a portion of therepetitive DNA segment in M. tuberculosis is used as target material foramplification by polymerase chain reactions, wherein amplificationproduces a 123 base-pair product.

In a further embodiment, a repetitive DNA segment in M. tuberculosis isused as a hybridization probe for directly detecting isolates of the M.tuberculosis complex in clinical material.

Additional features and advantages of the present invention are furtherdescribed, and will be apparent from the detailed description from thepresently preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a restriction map of λKE55 and λKE58, clones isolatedfrom a lambda phage library of Mycobacterium tuberculosis DNA. The mapis based on restriction digests of the plasmid subclones illustrated.Contiguous and overlapping regions are shown.

FIG. 2 illustrates a restriction endonuclease map of the repetitive DNAsegment in Mycobacterium tuberculosis. The segment was derived fromdigests of the 3 contiguous cloned Sal1 fragments found in λKE55 andλKE58. The location of the DNA sequence, which was selected as a targetfor amplification using polymerase chain reactions, is also shown.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention pertains to a composition comprising a repetitivesegment of Mycobacterium tuberculosis DNA that has been isolated,cloned, restriction mapped, and partially sequenced. The presentinvention further contemplates the usefulness of this segment as adiagnostic tool in detecting the presence of M. tuberculosis in clinicalmaterial.

Pursuant to the present invention, M. tuberculosis DNA segments wereisolated in purified form, inserted into a bacteriophage vector,followed by transfection of appropriate host cells. The resultingrecombinants or clones were radiolabeled and screened for use as M.tuberculosis-specific nucleotide probes. To this end, Southern and slotblots containing purified DNA from representative strains of the M.tuberculosis complex were hybridized with the labeled DNA of therecombinants. Representative strains of the M. tuberculosis complexinclude M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis, andclinical isolates of M. tuberculosis.

As will be shown in the following examples, three cloned DNA segments ofM. tuberculosis hybridized with multiple chromosomal fragments of M.tuberculosis complex, indicating the repetitive nature of the DNAsegments. Specifically, each segment was found to repeat in the range of10-16 times in the M. tuberculosis chromosome.

Since one of these cloned DNA segments represented only a small portionof the entire repetitive sequence, additional cloning was performed toisolate the complete repetitive segment. A lambda phage library wasestablished and two clones, λKE55 and λKE58, were found to contain therepetitive DNA fragment. The fragments of these 2 clones were subcloned,and the subclones were subsequently mapped with restrictionendonucleases.

Subsequent studies revealed that the repetitive DNA segment wasconserved in multiples copies in the chromosomes of individual strainsof M. tuberculosis complex. Further, the hybridization assaysdemonstrated the specificity of the repetitive segment for strains ofthe M. tuberculosis complex.

Additional studies demonstrated the various uses of the repetitive DNAsegment as a hybridization probe. Examples include direct detection ofM. tuberculosis in clinical samples, culture confirmation of isolatedstrains, and chromosomal fingerprinting methods.

A representative portion of the repetitive DNA segment in M.tuberculosis was subsequently sequenced. A 123 base-pair segment wasselected as a target for amplification using polymerase chain reactions,sometimes referred to hereinafter as "PCR". Amplification of the segmentusing PCR demonstrated the presence of a 123 base-pair segment in thechromosome of M. tuberculosis, and was specific for DNA from M.tuberculosis complex strains. Further, the sensitivity of detection byamplifying M. tuberculosis DNA may provide the basis for an assay todetect the organism directly in clinical material.

By way of example, and not limitation, the following examples serve tofurther illustrate the present invention in its preferred embodiments.

EXAMPLE 1 (1) Materials

Clinical isolates of M. tuberculosis and M. kansasii were obtained fromthe microbiology laboratories of McClellan Memorial Veterans Hospitaland Baptist Medical Center, Little Rock, Ark. M, avium strains LR541,542, 551, 552, 588, and 593 were AIDS-associated isolates obtained fromthe Centers for Disease Control, Atlanta, Ga. All other strains wereobtained from the reference laboratories at National Jewish Center forImmunology and Respiratory Medicine, Denver, Colo. The strains used arelisted in Table 1.

                  TABLE 1                                                         ______________________________________                                        Mycobacterial strains                                                         Organism, strain            Organism,                                         serotype    Organism, strain                                                                              strain, serotype                                  ______________________________________                                        M. tuberculosis                                                                           M. kansasii     M. avium complex                                  Clinical T2 TMC1204         LR147.1                                           H37Rv (TMC102)                                                                            TMC1217         LR588.1                                           H37Ra Goldman                                                                             Clinical K4     LR593.1                                           Clinical Tl Clinical K5     LR113.4                                           Clinical T3 Other           LR541.4                                           Clinical T4 M. fortuitum TMC1530                                                                          LR542.4                                           Clinical T5 M. simiae TMC1226                                                                             LR150.8                                           Clinical T6 M. gordonae TMC1324                                                                           LR551.8                                           M. bovis    M. chelonei TMC1543                                                                           LR552.8                                           TMC401                      TMC715.2                                          TMC410                      LR107.3                                           BCG Glaxo                   LR163.6                                           (TMC1024)                                                                     M. scrofulaceum             TMC1479.9                                         TMC1315.41                  LR158.10                                          TMC1309.42                  LR105.11                                          LR121.43                    TMC1466.13                                                                    LR127.19                                                                      LR141.26                                          ______________________________________                                    

(2) Preparation of Mycobacterial DNA

In order to isolate the DNA sequence that is specific for M.tuberculosis, the DNA must be separated from the rest of the cell. Toaccomplish this, cells were cultured in 100 ml of Middlebrook 7H9 broth(Difco Laboratories) containing 0.05% Tween 80 in 500 ml baffledside-arm flasks. Cultures were incubated at 37° C. on a rotary shaker. AKlett colorimeter was used to monitor the cell density. When thecultures were in log phase, D-cycloserine was added to a finalconcentration of one mg/ml. Cultures were reincubated until the densitydecreased slightly, which usually occurred within 48 to 72 hours. Cellswere harvested by centrifugation, washed once with: STE, and suspendedin 2 ml of lysis buffer containing 15% sucrose, 0.05M TRIS-hydrochloride(pH 8.5), and 0.05M EDTA. Lysozyme was added (1 mg/ml), and the cellsuspension was incubated for 30 minutes at 37° C. Proteinase K was added(0.1 mg/ml), and the suspension was incubated at 25° C. for 10 minutes.

Sodium dodecyl sulfate (SDS), a lysing agent, was added to a finalconcentration of 1%. Addition of the lysing agent resulted in the M.tuberculosis chromosomal DNA being accessible for subsequentpurification. For purification of the organism's chromosomal DNA, theDNA was extracted twice with phenolchloroform-isoamyl alcohol, purifiedby dialysis, and concentrated by ethanol precipitation. The DNAconcentration was determined by optical density at 260 nm.

The purified chromosomal DNA was then digested into fragments with arestriction endonuclease. Reaction mixtures contained 1 to 2 μg of DNAand 4 to 10 U of enzyme. Digest conditions were specified by themanufacturer of the enzyme (Bethesda Research Laboratory, Inc. or NewEngland BioLabs, Inc.). To verify that the digestion had proceededsufficiently for cloning, agarose gel electrophoresis was utilized tomeasure the size of the DNA fragments.

(3) Cloning

Each of the chromosomal DNA fragments was then inserted into a vector(e.g. bacteriophage) to form a recombinant molecule. To accomplish this,DNA from a clinical isolate of M. tuberculosis, designated T2, wasdigested with Mbol and ligated with a BamH1 digest of M13mp18replicative form. The recombinant molecule was then transfected intoEscherichia coli JM101 cells and plated. The recombinants, whichappeared as colorless plaques, were picked to freshly seeded agarplates. Duplicate plaque lifts were made from each plate.

Recombinant phage were propagated in log-phase cultures of E. coliJM101. The single-stranded DNA was isolated from culture supernatants bypolyethylene glycol precipitation. Double-stranded (replicative form)DNA was isolated from cell pellets by alkaline lysis method. To excisethe insert DNA, the replicative-form DNA was digested with EcoR1 andHind 111. The sizes of the excised fragments were determined by agarosegel electrophoresis.

(4) Labeling DNA Probes

To radiolabel the DNA probe, the DNA was labeled with [α-³² P]dCTP bynick translation. Single-stranded M13 DNA was labeled with ³² P byprimer extension, using the procedure of Hu and Messing (Gene17:271-277, 1982). Specific activity of the probe was about 10⁸ cpm/μgof DNA, and approximately 10⁶ to 10⁷ cpm was used in each hybridization.

(5) Slot Blot Hybridizations

To assess the potential of these recombinants for use as M.tuberculosis-specific probes, slot blot hybridizations were performed.Slot blots containing 128 ng of purified DNA from representative strainsof the M. tuberculosis complex (M. tuberculosis H37Rv, M, tuberculosisH37Ra, M bovis, and clinical isolates of M. tuberculosis) werehybridized with the labeled single-stranded form of the recombinants. Toaccomplish this, purified DNA was denatured in 0.4N NaOH at roomtemperature for 10 minutes, neutralized with an equal volume of 2Mammonium acetate (pH 7), and then loaded on a slot blotter (Minifold II;Schleicher and Schuell, Inc.) containing a BA85 nitrocellulose membrane.Next, fixing of the DNA to the membrane was done to prevent the DNA frombeing washed off the membrane during the subsequent hybridization/washstep. Fixing was carried out by placing the membranes in a vacuum ovenat 80° C.

The DNA was then blocked to prevent any non-specific binding to themembrane by the recombinant DNA and the labeled probe. Blocking wascarried out by incubating the membrane in a standard hybridizationsolution consisting of 6×SSC (0.9M NaCl plus 0.09M sodium citrate),5×Denhardt solution and 100 μg of denatured salmon sperm DNA per ml.EDTA (0.01M) and labeled probe DNA were added to the hybridizationsolution. Membranes were hybridized over night at 68° C. and then washedas follows: 2×SSC in 0.5% SDS at room temperature for 5 minutes, 2×SSCand 0.1% SDS at room temperature for 10 minutes, and 0.1×SSC and 0.5%SDS at 68° C. for 2.5 hours.

Membranes were exposed for variable lengths of time at -70° C. to x-rayfilm to detect blots where the ³² P-labeled DNA hybridized to M.tuberculosis complex DNA.

With the slot blot hybridizations, three recombinants, designatedM13KE37, M13KE49, and M13KE115, hybridized strongly with all strains andappeared to be good candidates for clinical probes. To accessspecificity, slot blots of DNA from mycobacteria commonly found insputum were hybridized with these three probes. No significanthybridization occurred with the DNA from clinical isolates and referencestrains of M. kansasii or with reference strains of M. fortuitum, M.chelonei, M. gordonae and M. simiae. Further, no significanthybridization was detected with clinical and reference strains of M.avium serotypes 1, 4, and 8 or with other reference strainsrepresentative of this species. When slot blots containing two-folddilutions of M. tuberculosis DNA were hybridized with these probes, 2 to4 ng of DNA was detected.

(6) Southern Blot Hybridizations

Another procedure, Southern blot hybridizations, was also used toascertain more specific information. The M13 recombinants, as previouslydescribed, were hybridized with membranes containing BamH1 digests ofDNA from members of the tuberculosis complex. Digests wereelectrophoresed on 0.8% agarose gels containing ethidium bromide andphotographed.

The DNA fragments were denatured and transferred to a GeneScreen Plusmembrane (DuPont, NEN Research Products) by using either a modifiedSouthern transfer method as described by Maniatis et al., (MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1982) or the alkaline transfer method of Chomczynski andQasba (Biochem. Biophys. Res. Commun. 122: 340-344, 1984). GeneScreenPlus was hybridized according to the manufacturer's instructions, exceptthat hybridization was performed at 68° C. without dextran sulfate. Themembranes were washed twice with 2×SSC at room temperature for 5minutes, twice with 2×SSC and 0.1% SDS at 68° C. for 30 minutes, andtwice with 0.1×SSC at room temperature for 30 minutes. The membraneswere then exposed for various lengths of time to x-ray film at -70° C.

Most notably, all three recombinants hybridized strongly with multipleDNA fragments of M. tuberculosis. M13KE37 hybridized strongly with 16BamH1 fragments of T2 DNA and with 12 fragments of H37Rv. Further,M13KE37 hybridized with nine fragments in the M. tuberculosis T1 digest(another clinical isolate) and with five in the M. bovis digest. Thefragments ranged from 18 to 1 kilobase pairs, indicating a repetitivesequence in the DNA of strains of the M. tuberculosis complex. However,the results also suggest that this sequence does not occur with the samefrequency in all strains.

A second recombinant, M13KE49, hybridized with 10 BamH1 fragments in thedigests of T2 DNA. Similarities in band patterns among the M.tuberculosis complex strains indicate that the DNA segment is conservedamong the complex strains.

Similarly, M13KE115 hybridized strongly with nine fragments in the T2digest and less with seven others. The band patterns of all the complexstrains were almost identical, indicating that the sequence is highlyconserved in all the strains. Further, the three recombinants did nothybridize to a significant extent with DNA from non-tuberculousmycobacteria.

The repetitive DNA segments, as observed among three cloned DNA segmentsof M. tuberculosis, should be useful in enhancing the sensitivity ofdetection for M. tuberculosis in clinical material by a factor of 10-16as compared to a DNA segment that occurs only once in the chromosome. Asa clinical probe in a direct hybridization assay, a repetitive segmentcould effectively shorten the time for diagnosing tuberculosis inclinical materials.

Additional studies were conducted with M13KE37 to isolate andcharacterize the complete repetitive sequence of the DNA segment fromMycobacterium tuberculosis. In addition, a portion of the DNA segmentwas selected as a target for amplification by polymerase chain reaction(PCR), a technique that greatly enhances the sensitivity of detection ofspecific DNA sequences.

EXAMPLE 2 1. Isolation of Subclones Containing the Repetitive Element

Since the repetitive fragment in M13KE37 is only a small portion of alarger repetitive sequence and contains fragments of nonrepetitive DNA,the following approach was used to isolate the complete repetitivesegment. A lambda phage library of M. tuberculosis DNA was establishedby cloning a partial EcoR1 digest of strain T2 into the EcoR1 site ofthe bacteriophage lambda EMBL3 (Frishauf AM, Lebrach H, Poustka A,Murray N. J Mol Biol 170:826, 1983). The phage were grown on E. colistrain Q359. Recombinant phage were identified by hybridizing plaquelifts with ³² P-labeled M13KE37. Two of the clones which were isolatedfrom the library, λKE55 and λKE58, were found to contain 400-bp Sal1fragments which were found to hybridize with one another, and 2 flankingSal1 sequences which partially hybridized one to another. Thesefragments of λKE55 and λKE58 were subcloned into the Sal1 site of pUC19(Yanisch-Perron C, Vieria J, and Messing J. Gene 33:103, 1985). Thesubclones were designated pDC51 (λKE55), pDC38 (λKE58), pDC22 (λKE55),pDC61 (λKE58), pDC2 (λKE55), pDC26 (λKE58), pDC106 (pDC51), pDC73(pDC22), and pDC92 (λKE55).

2. Restriction Enzyme Mapping of Subclones

The subclones were mapped with restriction endonucleases to verifycommon segments, that is fragments which share the repetitive element.As illustrated in FIG. 1, common sites for Sst11, Sma1, and Dra1 werefound in pDC51 and pDC38. A common BamH1 site was found in pDC22 andpDC61, and a common site for Xhol is found in pDC2 and pDC26. Usefuladditional clones include pDC106, pDC73, and pDC92, which containvarious portions of the repetitive sequence which have been used ashybridization probes. While 2 of the Sal1 clones (pDC22 and pDC16)contain a 400-bp Sal1 fragment of the repetitive unit, the other 4clones (pDC51, pDC2, pDC38, pDC26) contain in addition to the repetitiveelement nonrepetitive flanking sequences.

A restriction endonuclease map of the repetitive element was derivedfrom digests of the 3 contiguous cloned Sal1 fragments found in λKE55and λKE58. The location of the DNA sequence, which was selected as atarget for amplification using PCR, is shown in FIG. 2.

3. Conservation of the Repetitive Element

The question addressed herein was whether all of the copies found in theDNA of a single strain and in various strains of M. tuberculosis and M.bovis are identical. To this end, genomic DNA from M. tuberculosisstrains H₃₇ Rv, H₃₇ Ra Goldman, six clinical isolates, and three strainsof M. bovis were restricted with enzymes that cleave within theinsertion sequence, electrophoresed, blotted, and probed with ³²P-labeled Sal1-BamH1 or Sstl1 fragments. A single fragment of identicalsize (600 bp) was detected by hybridization in all strains, indicatingconservation of the Sst11 sites in all copies of the repeat in allstrains tested. Similarly, single bands of a size consistent with therestriction map were detected in all strains in which the DNA wasdigested with Sal1 (400 bp) and combinations of Xhol and Sma1 (1 Kb),Xho1 and BamH1 (400 bp), and Dra1 and Xho1 (950 bp).

The data demonstrated that 8 restriction sites for 6 restrictionendonucleases are conserved within multiple copies of the repetitiveelement in a single strain, across strain and across species lines.Within the limit of resolution of agarose gels the length of restrictionfragments formed by cleavage of two sites within the repeat unit(cleaved by the same enzyme or by combinations of enzymes) producesfragments of identical length. This indicates that the sequencesseparating the restriction sites are probably of identical or nearlyidentical sequence.

4. Specificity of the Repetitive Element

Southern blot hybridizations were performed with DNA from variousstrains of nontuberculous mycobacteria to confirm the specificity of therepeated sequence. When DNA of M. avium, M. intracellulare, M.scrofulaceum, M. Kansasii, M. gordonae, M. fortuitum or M. chelonei wasdigested with Sst11 or BamH1, enzymes which would produce single ormultiple hybridizing fragments respectively, and hybridized with probes,pDC106 and pDC73, no hybridization was noted. The data confirm that therepeat is not present in the DNA of mycobacteria outside of the M.tuberculosis complex. These results suggest that the sequence appearedat a time after the M. tuberculosis complex diverged from othermycobacteria.

5. Fingerprinting of Genomic DNA

When genomic DNA of M. tuberculosis was restricted with enzymes forwhich there were no sites in the repeat, multiple fragments ranging innumber from six to twelve were observed. Although we observeddifferences in patterns among strains, the fragments were too large tobe well resolved on the gels. In order to produce smaller fragments andto increase their number, an enzyme with a single restriction site inthe insertion sequence (BamH1) was chosen. Restriction with BamH1produces fragments which extend from that site to BamH1 sites (left andright) in the DNA which flanks the repetitive:element. The DNA extendingfrom the conserved BamH1 site in the repeat to the proximal BamH1 in theDNA flanking the left end of the repeat can be detected by hybridizationwith the Sst11 fragment of pDC106 (see FIG. 1). DNA extending from theBamH1 fragment in the repeat to the BamH1 site in the DNA flanking theright end of the repeat can be detected by hybridization with theBamH1 - Sal1 fragment of pDC73 (FIG. 1).

Southern blots of BamH1 restricted DNA from M. tuberculosis H37Rv andthe six clinical isolates demonstrate about 12 bands with each probeconfirming that there are multiple copies of the repetitive segment. Asexpected, the banding patterns for each strain obtained with the twoprobes differ from one another. While some of the fragments from thevarious strains of M. tuberculosis DNA hybridized with the same probeappear to be the same, each strain has a pattern which is characteristicof that strain and differs from the others. Such differences areapparent in Southern blots hybridized with either probe. The patternssuggest a relatedness between H37Rv and clinical isolates T2 and T4 incontrast to clinical isolates T1 and T5 which differ strikingly. H37Rvand H37Ra Goldman, virulent and avirulent derivatives of H37, were alsocompared. Even these two closely related strains exhibit significantdifferences in restriction fragments.

Differences in restriction sites flanking the repetitive sequence areuseful in characterizing the DNA of various M. bovis and M. tuberculosisstrains. The inventors found BamH1 to be useful for this purpose,because the fragments produced are well resolved on agarose gels.Further, by using the two cloned fragments (the Sst11 fragment and theBamH1-Sal1 fragment), two sets of 10-12 bands can be demonstrated in asingle digest. Other enzymes or combinations of enzymes may proveequally useful.

The use of the repetitive element in fingerprinting the DNA ofindividual strains should prove important in epidemiological studies.This approach is being applied to a number of bacterial pathogens. Owen,J. Med. Microbiol. 30: 89-99, 1989. In examining the DNA of only eightstrains, no identical patterns were detected. Although the rate at whichthe sequences diverge in M. tuberculosis in unknown, it is contemplatedthat the fingerprint patterns will be relatively stable, and thereforeuseful in studying various isolates from specific outbreaks oftuberculosis. More importantly, these results suggest that as morefingerprint patterns are studied, it will be possible to divide M.tuberculosis strains into subgroups based on overall patterns ratherthan specific band differences. For example, H37Rv, H37Ra, strain T2 andT4 are not identical, but may represent a subgroup distinct from strainsT1 and T5. These groupings may correlate with geographic origin,relative virulence or other variables.

EXAMPLE 3 (1) DNA Sequencing

DNA sequence analysis was conducted on a portion of the repetitive DNAsegment from Mycobacterium tuberculosis. Mycobacterial DNA was isolatedaccording to the procedure in Example 1. The concentration of DNA wasdetermined by measuring the optical density at 260 nm. A single fragmentof clone M13KE39 was obtained by partial digestion with endonuclease Sau3A and subcloned into M13mp18 and M13mp19 for sequencing using thedideoxy chain termination technique. Both strands were sequenced. Thesesubclones hybridized with the λKE55 and λKE58 clones described inExample 2.

A portion of the repetitive DNA segment from M. tuberculosis isillustrated in Table 2. As shown, the segment is comprised of 123base-pairs and a 6 base-pair internal Sal1 site. This sequence is partof a larger repetitive segment. On the basis of the analysis of thesequence of M13KE39, it was predicted that there would be a Sal1 sitelocated 55 bp from one end of the 123-bp amplified fragment. When theamplified fragment was digested with Sal1, it was cut into 2 fragmentswhich measure approximately 50 and 70 nucleotides in length. Thislocates the Sal11 site at the expected distance from the BamH1 site inthe repetitive sequence. The location of this specific sequence is shownon the restriction map of the repetitive element as illustrated in FIG.2.

                                      TABLE 2                                     __________________________________________________________________________    Sequence of the Repetitive DNA Segment from                                   Mycobacterium tuberculosis                                                    __________________________________________________________________________     ##STR1##                                            60                        ##STR2##                                           120                        ##STR3##                                           123                       __________________________________________________________________________

The 20 base regions used for priming the reaction are underlined.

(2) Polymerase Chain Reactions

A portion of the repetitive DNA segment from Mycobacterium tuberculosisas shown in Table 2 was selected as a target for amplification usingpolymerase chain reaction (PCR).

Nucleotide primers were synthesized on a DNA synthesizer (Model 381A,Applied Biosystems, Foster City, Calif.) using B-cyanoethylphosphoramidite chemistry. The DNA was deblocked using ammoniumhydroxide and purified by passage through a Sephadex G-25 column(NAP-25, Pharmacia, Piscataway, N.J.), and ethanol precipitation. Thesequences of the oligonucleotide primers were 5'-CCTGCGAGCGTAGGCGTCGG-3'and 5'-CTCGTCCAGCGCCGCTTCGG-3'. Nucleotide primers corresponding to theindicated sequence were synthesized for use in PCR.

Amplification reactions were performed using Thermus aquaticus (Taq)polymerase and reagents according to the manufacturer's instructions(GeneAmp Kit; Perkin-Elmer Cetus). Buffer, nucleotides, primers (1 μMeach), and enzymes were mixed and then dispensed in 45 μl aliquots. DNAwas added in a volume of 5 μl, and the reaction mixture was overlayedwith mineral oil. A control tube containing no DNA was included witheach set of reactions.

The reaction was performed using an automated thermal cycler (DNAThermal Cycler; Perkin-Elmer Cetus). The samples were denatured at 94°C. for five minutes. Twenty-five amplification cycles were performed. Acycle consisted of denaturation at 94° C. for two minutes, annealing ofprimers at 68° C. for two minutes, and primer extension at 72° C. fortwo minutes. The extension time was increased by 5 s with eachsubsequent cycle. The product was analyzed by electrophoresis on 0.8%agarose gels with 12% acrylamide gels. The DNA was stained with ethidiumbromide and photographed on a 302-nm ultraviolet transilluminator.

Amplification of the sequence of the repetitive DNA segment from M.tuberculosis produces a 123 base-pair segment with a 6 base-pairinternal site for endonuclease Sal1. An annealing temperature of 68° C.is necessary to produce the segment. The results further indicate thatthe primer segments have a 75% G+C content with the A and T residueswell spaced, which allows for the use of a high primer annealingtemperature. This contributes to the specificity of the reaction and mayalso increase the sensitivity of the reaction.

Although hybridization assays demonstrated the repetitive nature of theDNA sequence in M. tuberculosis, it was not known if each copy of thesequence was conserved or if each could be amplified using theoligonucleotide primers. To this end, DNA from M. tuberculosis strain T2was digested with endonuclease BamH1, which does not cleave within thePCR target. Two aliquots containing 1.45 μg and 145 ng of DNA wereelectrophoresed. The lane containing 1.45 μg of DNA was transferred to anylon membrane (GeneScreen Plus), using an alkaline procedure.Chomczynski, Biochem. Biophys. Res. Commun. 122: 340-344, 1984. Thatlane was further blotted and probed by hybridization with labeled PCRproduct, the 123 base-pair segment produced by the PCR reaction. Thelane containing 145 ng of DNA was sliced into 28 fractions, melted, andthen diluted 1000-fold in water. 5 μl aliquots of certain fractions werethen amplified by PCR.

The results indicate that at least 12 copies of the sequence existed.Further, all of the individual copies were amplified using the sameprimers.

Next, the specificity of the PCR was determined. Accordingly, DNA from39 strains of mycobacteria were assayed. Relatively high amounts ofinput DNA (625 pg per reaction) were used to ensure that anamplification would be detected.

DNA was amplified from 10 strains of M. tuberculosis and M. bovis and 1strain of M. simiae to yield the 123 base-pair product. Also, when theamplification products were digested with Sal1, identical Sal1 fragmentswere obtained with these strains. On the other hand, no product wasdetected with the DNA from nontuberculosis mycobacteria (e.g., 28strains of the M. avium complex, M. kansasii, M. scrofulaceum, M.fortuitum, M. chelone; and M. gordonae). Lack of amplification wasconfirmed by hybridization of a blot of the gel.

Finally, the sensitivity of detection of DNA by PCR was assessed.Ten-fold serial dilutions of M. tuberculosis strain T2 DNA wereamplified. One set of samples was amplified for 25 cycles and anotherfor 30 cycles. The PCR product was detected after 25 cycles from 100 fgof input DNA, while detection occurred after 30 cycles with only 1 fg ofinput DNA (or roughly equivalent to one copy of the M. tuberculosischromosome). No product was detected after 30 cycles with M. avium DNA,human DNA alone, or in the control sample containing no input DNA.

These results demonstrate that amplification of the target DNA sequencein M. tuberculosis can provide a diagnostic approach for dramaticallyincreasing the sensitivity of detection of the organism directly inclinical material. Although only one part of the entire repetitive DNAsequence was amplified, it is contemplated that any part of the entiresequence can be similarly used.

(3) Detection of M. tuberculosis in Sputum Samples Using PCR

Techniques were developed that enable the PCR to be applied directly toclinical samples. Procedures involved establishing a method for lysingmycobacteria, extracting the DNA, and testing for the presence of the123-bp fragment by PCR. 162 sputum samples, half of which were smearpositive for acid-fast bacilli, were tested using this technique.

(a) Patient samples. sputum samples were obtained from the ArkansasState Health Department laboratory where the specimens were treated withthe standard protocol of n-acetylcysteine-NaOH. The sediments remainingafter microscopy and inoculation of solid media were stored at 4° C.until they were processed for PCR.

(b) Specimen processing for PCR. Sediment from the sputum sample wascentrifuged in a screw cap microfuge tube for 5 minutes. The pellet wassuspended in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) containing 20mg/ml lysozyme and incubated at 37° C. for 2 hours. Subsequently, NaOHand sodium dodecyl sulfate were added to final concentrations of 0.5Nand 1% respectively, and the tube was placed in a boiling water bath for5 minutes. Once at room temperature the sample was neutralized with HCl.The DNA was extracted and concentrated by binding to powdered glassusing the GeneClean kit (B10 101 Inc., La Jolla, Calif.). The DNA waseluted from the powdered glass in 10 ml of water.

(c) Controls. As a control for the lysis reagents and procedure, a tubecontaining 10³ M. tuberculosis organisms and a tube containing noorganisms were processed in each batch of clinical samples. A positivecontrol tube containing 100 pg of M. tuberculosis DNA and a negativecontrol tube containing no DNA were included with each set of reactions.Control DNA that produces a 600-bp PCR product with the same primers wasincluded in each reaction as an internal control. The control DNA wasconstructed as follows.

A single-stranded 46 nucleotide oligomer was synthesized which consistsof the two, 20 base primer sequences with a 6 base EcoR1 restrictionenzyme site in the middle. The DNA was synthesized by Bio-Synthesis,Inc. (Denton, Tex.) and was purified by polyacrylamide gelelectrophoresis. The 46mer was amplified by PCR, and the double-strandedproduct was blunt-end ligated into the Sma1 site of a modified pUC 19vector from which the EcoR1 site had been removed. DNA from theresulting clone was digested with EcoR1 and ligated with a 550-bp EcoR1fragment of DNA isolated from Salmonella typhimurium. This clone, whichcontains a fragment of S. typhimurium DNA inserted between the primers,is referred to as pDC139.

(d) Results. For the clinical trial 162 sputum samples were processedfor PCR testing according to the procedure previously described.Following amplification, the product was detected by acrylamide gelelectrophoresis. Samples were determined to be PCR positive based on thevisualization of a 123-bp fragment on the gel, and negative if a 123-bpfragment was absent. The 600-bp fragment of the internal control DNA wasalso present.

Of the 162 sputum samples tested, 82 were smear positive for acid-fastbacilli. Of the 94 specimens from patients diagnosed as having pulmonarytuberculosis, 51 were culture positive, smear positive, or both. Fiftyof these tested PCR positive. Of the 42 specimens from patients withnontuberculosis mycobacterial disease, 41 tested PCR negative. All 26specimens from patients without mycobacterial infection were PCRnegative.

This assay provides a sensitive and specific means for the laboratorydiagnosis of tuberculosis within a brief period of time (48 hours).Further, the assay is relatively simple to perform, providing additionalbenefits to health care institutions in their delivery of diagnosticservices.

(4) Detecting M. tuberculosis in Sputum Samples Using a Probe Assay

A simple and efficient procedure for lysis of mycobacteria from sputumwas developed for the application to a membrane-based hybridizationassay. Initially, pure cultures of mycobacteria were used for testingthe lysis procedure and subsequently processed sputum sediment was used.Optimum lysis was achieved with the following procedure.

A cell suspension or sputum sediment was centrifuged at 12,000×g for 5minutes. The supernatant was decanted, and the cell pellet wasresuspended in 500 μl TE buffer. 100 mg of zirconium beads (0.1 mm) wasadded, and the tube was agitated at high speed in a Minibead Beater(Biospec Products, Bartelsville, Okla.) for 3 minutes. The tube was thenplaced in a boiling water bath for 10 minutes to render the samplenoninfectious. The sample was denatured by adding NaOH to the finalconcentration of 0.25N NaOH. The tube was centrifuged for 2 minutes toremove debris, and the entire sample was loaded on a membrane using aslot blotter apparatus. The membranes were hybridized and washed toremove unbound probe, according to the conditions as described inExample 1.

Using ³² P-labeled pDC92 (described in Example 2), to probe themembranes, as few as 10² -10³ mycobacterial cells per ml were detected.Since a limited number of smear positive and smear-negative sputumsamples have been processed with this procedure, the sensitivity andspecificity have not yet been determined.

The present invention involves the various embodiments associated with acomposition comprising a repetitive segment of Mycobacteriumtuberculosis DNA and its use in all respects, and is not to be construedas limited to any specific aspect or embodiment except as defined by thelawful scope of the appended claims.

We claim:
 1. A composition used as target material for amplification bypolymerase chain reaction, consisting of:a repetitive DNA segment in thechromosome of M. tuberculosis that is specific for M. tuberculosiscomplex strains, wherein the segment consists of a 123 base-pair DNA(nucleotide) sequence: ##STR4##
 2. The composition of claim 1, whereinthe nucleotide primers for the DNA sequence consists of5'-CCTGCGAGCGTAGGCGTCGG-3' and 5'-CTCGTCCAGCGCCGCTTCGG-3'.
 3. Thecomposition of claim 1, wherein the DNA sequence contains an internalsite for endonuclease Sal1 consisting of: 5'-GTCGAC-3'.
 4. Thecomposition of claim 1, wherein amplification of the DNA segmentproduces a 123 base-pair product.
 5. A method of diagnosing tuberculosisin clinical material, comprising the steps of:amplifying a repetitiveDNA segment of Mycobacterium tuberculosis that is specific for M.tuberculosis complex strains using polymerase chain reaction, whereinthe segment consists of a 123 base-pair DNA (nucleotide) sequence:##STR5## detecting the presence of the 123 base-pair DNA sequence. 6.The method of claim 5, wherein the nucleotide primers for the DNAsequence consists of: 5'-CCTGCGAGCGTAGGCGTCGG-3' and5'-CTCGTCCAGCGCCGCTTCGG-3'.
 7. The method of claim 5, wherein the DNAsequence contains an internal site for endonuclease Sal1 Aconsisting of:5'-GTCGAC-3'.
 8. The method of claim 5, wherein amplification of the DNAsegment produces a 123 base-pair product.
 9. A method for directlydetecting Mycobacterium tuberculosis in clinical samples usingpolymerase chain reaction, comprising the steps of:(a) lysingmycobacteria in clinical samples; (b) extracting the DNA of themycobacteria; (c) assaying the DNA for the presence of a DNA fragment bypolymerase chain reaction, wherein the DNA fragment consists of a 123base-pair DNA (nucleotide) sequence: ##STR6## (d) detecting the presenceof the 123 base-pair DNA sequence.
 10. The method of claim 9, whereinthe clinical samples are sputum samples.
 11. The method of claim 9,wherein the nucleotide primers for the DNA sequence consists of:5'-CCTGCGAGCGTAGGCGTCGG-3' and 5'-CTCGTCCAGCGCCGCTTCGG-3'.
 12. A DNAsegment, pDC139, for use as an internal control in polymerase chainreaction for the detection of Mycobacterium tuberculosis in clinicalsamples.
 13. The DNA segment of claim 12, wherein amplification of theDNA segment produces an approximate 600-bp product.
 14. The DNA segmentof claim 12, wherein the nucleotide primers for the DNA segment consistsof: 5'-CCTGCGAGCGTAGGCGTCGG-3' and 5'-CTCGTCCAGCGCCGCTTCGG-3'.
 15. Amethod for confirming the amplification of DNA with polymerase chainreaction, comprising the steps of:(a) lysing microorganisms in clinicalsamples; (b) extracting the DNA of the microorganisms; (c) addingnucleotide primers to the samples of step (b); (d) adding a DNA segment,as an internal control, to the samples of step (c) which uses thenucleotide primers for amplification; (e) amplifying the microbial DNAand the internal control DNA segment by polymerase chain reaction; and(f) detecting the presence of at least one DNA fragment as defined bythe nucleotide primers in step (c).
 16. The method of claim 15, whereinthe internal control DNA segment is pDC139.
 17. The method of claim 15,wherein amplification of the internal control DNA segment producesapproximately a 600-bp product.
 18. The method of claim 15, wherein thenucleotide primers for the microbial DNA and the internal control DNAsegment consists of: 5'-CCTGCGAGCGTAGGCGTCGG-3' and5'-CTCGTCCAGCGCCGCTTCGG-3'.
 19. The method of claim 15, whereinamplification of the microbial DNA produces a 123 base-pair productconsisting of the DNA sequence: ##STR7##